The Characterization of the Mitochondrial Genome of Fulgoraria rupestris and Phylogenetic Considerations within the Neogastropoda

Fulgoraria rupestris is a predatory marine gastropod belonging to Neogastropoda and possessing considerable taxonomic significance. However, research on this species remains limited. We acquired the complete mitochondrial genome of F. rupestris through second-generation sequencing and conducted an analysis of its genome structural features. The mitochondrial genome of F. rupestris spans a total length of 16,223 bp and encompasses 37 genes (13 protein-coding genes (PCGs), 22 transfer RNAs, and 2 ribosomal RNAs). Notably, most tRNAs exhibit the typical cloverleaf structure, but there is an absence of the Dihydrouridine (DHU) arm in the trnS1 and trnS2 genes. The A + T content is 68.67%, indicating a pronounced AT bias. Additionally, we conducted a selection pressure analysis on the mitochondrial genomes of four species within Volutidae, revealing that all PCGs are subjected to purifying selection. In comparison to other species within Neogastropoda, F. rupestris shares an identical gene arrangement. Additionally, based on mitochondrial genome sequences of the 13 PCGs from 50 species within Neogastropoda, we constructed a phylogenetic tree. The phylogenetic tree indicates F. rupestris forms a clade with species within the family Volutidae (Cymbium olla, Neptuneopsis gilchristi, and Melo melo). This study serves as a valuable reference for future research on F. rupestris, offering insights for the upcoming phylogenetic and taxonomic classification within Neogastropoda. Furthermore, the findings provide valuable information for the development of genetic resources in this context.


Introduction
Neogastropoda, a taxonomically rich assemblage of marine predatory invertebrates, is systematically categorized into eight superfamilies: Buccinoidea, Conoidea, Mitroidea, Muricoidea, Olivoidea, Turbinelloidea, Volutoidea, and Pholidotomoidea (fossil only) [1,2].Since the Cretaceous period, Neogastropoda has undergone prolific diversification, colonized nearly all of the world's oceans, and asserted dominance within numerous shallow marine ecosystems [3,4].An integral trait of Neogastropoda is its prominent predatory behavior, with most of its constituent species exhibiting carnivorous feeding strategies.The evolution of this behavior is attributed to significant morphological adaptations, which encompass the elongation of the proboscis, the relocation of the mouth to the anterior end of the head, and the development of highly specialized tentacles [5][6][7][8].Given the extensive species diversity within Neogastropoda, its taxonomic classification has undergone significant modifications throughout its evolutionary history.The Neogastropoda are considered monophyletic in morphological classifications [9][10][11].The phylogenetic relationships among Neogastropod families are quite unstable and highly controversial in molecular analyses [12,13].The phylogenetic relationships among Neogastropod superfamilies remain unresolved [13].
Pilsbry et al. [19] initially classified Volutidae into 12 subfamilies [20].In their recent taxonomy, Bouchet et al. [16] further delineated Volutidae into ten subfamilies, with two subfamilies recognized as extinct.According to WoRMS (accessed on 15 July 2024), Volutidae is divided into 76 genus-level taxonomic units.Moreover, due to convergent morphological characteristics and the plasticity influenced by environmental factors, the taxonomy and phylogeny of Volutidae have been perplexing [21].When establishing classifications at levels such as families and genera, morphological features are susceptible to subjective interpretation by taxonomists.
Volutidae, a predatory marine gastropod within Neogastropoda [21], can trace its origins back to the late Early Cretaceous period [22][23][24].Most species from Volutidae inhabit shallow softbottom substrates in tropical and temperate regions, with certain derived species also distributed in polar and deep-sea areas [25].Many species within the Volutidae hold economic significance, being valuable components of luxury seafood and traditional medicines [26].Recently, there has been an increase in research on Volutidae [27][28][29][30][31][32][33][34][35][36][37].Fulgoraria rupestris, a species belonging to the Volutidae family (Neogastropoda: Volutoidea), exhibits a slightly hemispherical apex with an elliptical aperture.Its outer lip is distinguished by prominent thickness and distinctive wave-like features, often accompanied by brown markings on the outer wall [38].However, literature surveys reveal a relatively limited amount of research on F. rupestris.
The rapid advancement of molecular biology techniques has made significant contributions to resolving complex issues in morphological classification [39][40][41][42].Mitochondrial genes, known for their straightforward molecular structure, strict maternal inheritance, minimal recombination, and rapid evolutionary rate, have become valuable molecular markers.Particularly, complete mitochondrial genomes serve as excellent tools for studying phylogenetic relationships and taxonomic identification.Furthermore, compared to relying on individual mitochondrial genes, phylogenetic analysis based on the 13PCGs of mitochondrial genomes can enhance the resolution and statistical confidence of phylogenetic trees [43][44][45].
However, research on the complete mitochondrial genome of species within the Volutidae, including F. rupestris, is still limited.This study aims to elucidate the complete mitochondrial genome sequence of F. rupestris, analyze its basic nucleotide composition characteristics, explore interspecies evolutionary patterns, and construct a phylogenetic tree.We anticipate that the results of this study will markedly advance our understanding of the evolution and systematic classification within Neogastropoda, providing essential reference information for the development of genetic resources.

Sample Preparation and DNA Extraction
One sample of F. rupestris was collected in November 2018 from the sea area of Zhoushan, Zhejiang, China (30 • 19 ′ N, 122 • 72 ′ E), using the bottom trawl technique [46].Taxonomic specialists from the Museum of Marine Biology at Zhejiang Ocean University identified the specimen.Fresh tissues were dissected from the shell and after removing the digestive glands, the muscle tissues were stored in Ethanol absolute.Total DNA was extracted using the salt precipitation method [47].

Mitochondrial DNA Sequencing and Assembly
The mitochondrial genome is sequenced using the Illumina Novaseq TM platform at Shanghai Yuanshen Biomedical Technology Co. Ltd. (Shanghai, China) Initially, the genomic DNA of the samples undergoes quality control.Upon passing quality control, the DNA is fragmented into 300-500 base-pair fragments through ultrasonication, followed by purification.Subsequently, sequencing libraries are constructed from the fragmented DNA.The steps encompass DNA end repair, A−tailing at the 3 ′ end, the ligation of sequencing adapters, gel electrophoresis for the recovery of target fragments, the PCR amplification of target fragments, and, ultimately, the construction of sequencing libraries.Prior to sequencing, the constructed library undergoes quality control.Once it passes the quality check, sequencing is performed using the Illumina Novaseq TM platform.After obtaining raw data, filtering is carried out to exclude sequencing adapter sequences, low-quality reads, sequences with high N rates, and short-length sequences.This process results in high-quality sequencing data [48].The preliminary assembly results are obtained using GetOrganelle (https://github.com/Kinggerm/GetOrganelle/, accessed on 1 October 2023) and the best assembly results are achieved through multiple correction iterations.The assembly of the mitochondrial genes of F. rupestris is validated by BLAST against the cox1 barcode sequences in GenBank (https://www.ncbi.nlm.nih.gov/,accessed on 10 October 2023).

Genome Annotation and Bioinformatics Analysis
Genome annotation was conducted using the online tool MITOS (http://mitos2.bioinf.unileipzig.de/index.py,accessed on 6 January 2024) [49].The invertebrate mitochondrial genetic code was selected and the usage of start and stop codons was compared with those of closely related relatives [20,26].Following correction with Sequin, the complete mitochondrial genome data were uploaded to the NCBI database (https://www.ncbi.nlm.nih.gov/,accessed on 6 January 2024) to obtain the GenBank accession number.The circular mitochondrial genome map was generated using the online platform Proksee (https://proksee.ca/,accessed on 6 January 2024).DAMBE 7.0 [50] was employed to calculate the content of ATCG bases in the mitochondrial genome, as well as the content of the 13 protein-coding genes (PCGs).The skew values were computed using the formulas AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [51].MEGA X was used to calculate the frequency of amino acid usage and the relative synonymous codon usage (RSCU) in PCGs, while Ka/Ks (non-synonymous to synonymous substitutions) ratios were computed using DnaSP6.0[52,53].

Phylogenetic Analysis
We constructed a phylogenetic tree based on the mitochondrial genome sequences of 50 species within Neogastropoda, including the newly sequenced F. rupestris and members of 5 superfamilies (Volutoidea, Buccinoidea, Conoidea, Muricoidea, and Olivoidea) downloaded from the NCBI database (Table 1).Anodonta euscaphys and Anodonta arcaeformis were included as outgroups with GenBank accession numbers KP187851 and KF667530, respectively.The phylogenetic analysis was conducted using the Bayesian inference (BI) method with MrBayes 3.2.7aand the maximum likelihood (ML) method with IQ−tree 2.1.3[54,55].Firstly, the 13 protein-coding gene sequences from 50 species were combined into a FASTA file and aligned by codon using the ClustalW algorithm in MEGA X software [52].The aligned sequences were then trimmed, and the processed data were imported into the IQ-TREE program for analysis [55].A chi-square test was performed, followed by the utilization of ModelFinder to automatically compute and select the best substitution model (GTR + F + R7) for constructing the ML tree [56,57].Bayesian analysis was performed using MrBayes v3.2 [54], in conjunction with PAUP v4.0, Modeltest v3.7, and MrModeltest v2.3 from the MrMTgui v1.0 software.The best substitution model (GTR + I + G) was selected based on the AIC information criterion [58,59].The BI tree was constructed using the Markov Chain Monte Carlo (MCMC) sampling method, with sampling every 1000 generations.The analysis ran for a total of 8 million generations, with the initial 25% of sampled data discarded as burn-in.The resulting consensus tree was obtained, and Posterior Probabilities (PPs) were calculated.Finally, the phylogenetic tree was visualized and edited using FigTree v1.4.3 and Adobe Photoshop 2019.

Mitochondrial Genome Structural Features
The mitochondrial genome of F. rupestris has been deposited in NCBI with GenBank accession number OR588873.The mitochondrial genome sequence of F. rupestris exhibits a classic circular configuration, with a length of 16,223 bp, encompassing 37 genes (Figure 1).These genes include 13 PCGs, 22 tRNAs, and 2 rRNAs (12S rRNA and 16S rRNA).Among these genes, 29 genes are situated on the plus strand, while 8 genes are positioned on the minus strand (Figure 1, Table 2).The range of base pairs for the 13 PCGs is from 159 bp (atp8) to 1722 bp (nad5) (Table 2).In the mitochondrial genome of F. rupestris, the largest overlapping region is 23 bp between nad2 and cox1, while the maximum intergenic nucleotide region is 122 base pairs between trnE and 12S rRNA.

Mitochondrial Genome Structural Features
The mitochondrial genome of F. rupestris has been deposited in NCBI with GenBank accession number OR588873.The mitochondrial genome sequence of F. rupestris exhibits a classic circular configuration, with a length of 16,223 bp, encompassing 37 genes (Figure 1).These genes include 13 PCGs, 22 tRNAs, and 2 rRNAs (12S rRNA and 16S rRNA).Among these genes, 29 genes are situated on the plus strand, while 8 genes are positioned on the minus strand (Figure 1, Table 2).The range of base pairs for the 13 PCGs is from 159 bp (atp8) to 1722 bp (nad5) (Table 2).In the mitochondrial genome of F. rupestris, the largest overlapping region is 23 bp between nad2 and cox1, while the maximum intergenic nucleotide region is 122 base pairs between trnE and 12S rRNA.The mitochondrial genome of F. rupestris encompasses two rRNA genes, namely 12S rRNA, spanning 833 bp in length and 16S rRNA and measuring 1,366 bp.12S rRNA is situated between trnE and trnV, while the 16S rRNA gene is positioned between trnV and trnL (Table 2).The 22 tRNA genes collectively cover a sequence length of 1,490 bp, with individual lengths ranging from 65 to 70 bp.Notably, trnY, trnC, and trnQ each comprise 65 bp, while trnF and trnP each extend to 70 bp.Furthermore, trnL and trnS each possess two copies.Except for the absence of the DHU arm in trnS1 and trnS2, the remaining 20 tRNA genes exhibit the typical cloverleaf secondary structure (Figure 2).Intriguingly, trnL1 presents a U−U mismatch in the TΨC stem.Moreover, apart from trnH, trnP, and trnS, all other tRNA species display G−U mismatches.Specifically, trnA, trnE, trnG, trnK, trnL2, trnM, trnN, trnR, trnS2, trnT, trnV, and trnY exhibit G−U mismatches in the aminoacyl stem.Furthermore, trnD, trnE, trnF, trnG, trnL1, trnQ, trnR, and trnW display G−U mismatches in the DHC loop.Notably, trnA, trnK, trnN, trnS1, and trnW show G−U mismatches in the anticodon stem.

Nucleotide Composition and Base Skew Analysis
The nucleotide composition analysis of the mitochondrial genome in F. rupestris reveals distinct patterns.The respective percentages for each nucleotide are as follows: A at 30.55%, T at 38.12%, G at 16.01%, and C at 15.32% (Table 3).The cumulative A + T content stands at 68.67%, surpassing the G + C content of 31.33%.Notably, AT base pairs predominate, as evidenced by an AT-skew value of −0.11, indicating a subtle bias toward T, while the GC-skew value of 0.02 suggests a preference for G. Deeper insights emerge from the analysis of individual PCGs.The A content spans from 23.08% to 37.02%, T from 33.20% to 43.31%, G from 12.38% to 21.79%, and C from 11.94% to 17.57%.AT-skew values range from −0.26 to −0.05, while GC-skew values range from −0.13 to −0.20.Particularly noteworthy is the nad2 gene, exhibiting a notably low C content (11.94%) and a higher G content (18.06%), resulting in an elevated G-base skew rate of 0.20.

Nucleotide Composition and Base Skew Analysis
The nucleotide composition analysis of the mitochondrial genome in F. rupestris reveals distinct patterns.The respective percentages for each nucleotide are as follows: A at 30.55%, T at 38.12%, G at 16.01%, and C at 15.32% (Table 3).The cumulative A + T content stands at 68.67%, surpassing the G + C content of 31.33%.Notably, AT base pairs predominate, as evidenced by an AT-skew value of −0.11, indicating a subtle bias toward T, while the GC-skew value of 0.02 suggests a preference for G. Deeper insights emerge from the analysis of individual PCGs.The A content spans from 23.08% to 37.02%, T from 33.20% to 43.31%, G from 12.38% to 21.79%, and C from 11.94% to 17.57%.AT-skew Table 3. Base content in the mitogenome of F. rupestris.

Amino Acid Composition and Codon Usage
The analysis of amino acid content reveals that Leu1, Phe, Ile, and Tyr are the four most prevalent amino acids, constituting 11.40%, 8.90%, 7.11%, and 6.13%, respectively (Figure 3).According to the RSCU values of the 13 PCGs, it was found that UUA (Leu2), GCU (Ala), UCU (Pro), and AUU (Ile) are the most frequently used codons, with UUA at 2.22%, GCU at 1.96%, UCU at 1.71%, and AUU at 1.61% (Figure 4).The analysis of start and stop codons for PCGs indicates that, apart from nad2 and nad5, which commence with ATT, the rest of the genes initiate with ATG and terminate with TAA or TAG as stop codons.nad2 28. 24

Amino Acid Composition and Codon Usage
The analysis of amino acid content reveals that Leu1, Phe, Ile, and Tyr are the four most prevalent amino acids, constituting 11.40%, 8.90%, 7.11%, and 6.13%, respectively (Figure 3).According to the RSCU values of the 13 PCGs, it was found that UUA (Leu2), GCU (Ala), UCU (Pro), and AUU (Ile) are the most frequently used codons, with UUA at 2.22%, GCU at 1.96%, UCU at 1.71%, and AUU at 1.61% (Figure 4).The analysis of start and stop codons for PCGs indicates that, apart from nad2 and nad5, which commence with ATT, the rest of the genes initiate with ATG and terminate with TAA or TAG as stop codons.

Selection Pressure Analysis
We selected mitochondrial genomes of four species from Volutidae to analyze selection pressure.The calculated Ka/Ks values for all 13 PCGs are below 1 (Figure 5).Notably, atp8 exhibits the highest value at 0.38, while cox1 has the lowest value at 0.05.The overall Ka/Ks ratio below 1 implies that mutations have predominantly led to synonymous substitutions, indicating a purifying selection impact on Volutidae species throughout their evolutionary history.

Selection Pressure Analysis
We selected mitochondrial genomes of four species from Volutidae to analyze selection pressure.The calculated Ka/Ks values for all 13 PCGs are below 1 (Figure 5).Notably, atp8 exhibits the highest value at 0.38, while cox1 has the lowest value at 0.05.The overall Ka/Ks ratio below 1 implies that mutations have predominantly led to synonymous substitutions, indicating a purifying selection impact on Volutidae species throughout their evolutionary history.
We selected mitochondrial genomes of four species from Volutidae to analyze selection pressure.The calculated Ka/Ks values for all 13 PCGs are below 1 (Figure 5).Notably, atp8 exhibits the highest value at 0.38, while cox1 has the lowest value at 0.05.The overall Ka/Ks ratio below 1 implies that mutations have predominantly led to synonymous substitutions, indicating a purifying selection impact on Volutidae species throughout their evolutionary history.

Gene Order
Comparing the gene order of mitochondrial genomes of 50 species within Neogastropoda (Table 1), including F. rupestris, reveals that the gene order has changed in M. melo, N. gregarious, F. similis, G. moosai, and O. dimidiata, while the gene order is consistent among the remaining 44 species(Figure 6).M. melo and N. gregarius lack the trnF gene.F. similis underwent a gene inversion at trnS2−cob, resulting in the gene sequence becoming cob−trnS2.G. moosai presents a distinct order in the

Gene Order
Comparing the gene order of mitochondrial genomes of 50 species within Neogastropoda (Table 1), including F. rupestris, reveals that the gene order has changed in M. melo, N. gregarious, F. similis, G. moosai, and O. dimidiata, while the gene order is consistent among the remaining 44 species (Figure 6).M. melo and N. gregarius lack the trnF gene.

Phylogenetic Relationships
We performed a phylogenetic analysis on the 13 PCG sequences extracted from 50 species, encompassing 5 superfamilies (i.e., Volutoidea, Buccinoidea, Conoidea, Muricoidea, and Olivoidea) within the Neogastropoda.Based on two methods (ML and BI), nearly identical topologies were obtained.A. euscaphys and A. arcaeformis were chosen as outgroups in constructing the phylogenetic tree (Figure 7).
The consolidation of Neogastropoda as a monophyletic group received substantial support from robust statistical values.In this analysis, F. rupestris formed a highly supported clade alongside C. olla, N. gilchristi, and M. melo.Buccinoidea, Muricoidea, Volutoidea, and Olivoidea clustered together to form a branch (bootstrap probability of 0.7459).Within this branch, the bootstrap support value for the relationship among Muricoidea, Volutoidea, and Olivoidea is 0.6916.

Phylogenetic Relationships
We performed a phylogenetic analysis on the 13 PCG sequences extracted from 50 species, encompassing 5 superfamilies (i.e., Volutoidea, Buccinoidea, Conoidea, Muricoidea, and Olivoidea) within the Neogastropoda.Based on two methods (ML and BI), nearly identical topologies were obtained.A. euscaphys and A. arcaeformis were chosen as outgroups in constructing the phylogenetic tree (Figure 7).

Basic Features of the Mitogenome of F. rupestris
Fulgoraria rupestris, like most gastropods, possesses a mitochondrial genome consisting of 37 genes [60][61][62].The sequence lengths of the other three species (M.melo, N. gregarious, and C. olla) range from 15,312 to 15,721 base pairs.In the mitochondrial genome of F. rupestris, there is a D-loop region spanning 975 base pairs in length between trnF and cox3, which results in a total mitochondrial genome length of 16,223 bp in F. rupestris.The genome composition exhibits a pronounced AT bias, consistent with findings reported in previous studies [63][64][65].
Mitochondrial genes, with their high conservation, limited recombination, and maternal inheritance, are utilized to elucidate the evolutionary relationships among various animal taxa [66,67].In contrast to vertebrate mitochondrial genomes, those found in mollusks demonstrate notable heterogeneity in both length and structure.This variability is attributed to disparities in gene loss or duplication, as well as variations in the position and strand specificity of tRNA, protein-coding, and rRNA genes [68].In this study, among the four known species sequences within Volutidae, three exhibit a consistent gene order, while M. melo lacks trnF.This deletion in the non−coding region may be attributed to slippage events occurring in regions with high A/T or AT/TA repeats.The consolidation of Neogastropoda as a monophyletic group received substantial support from robust statistical values.In this analysis, F. rupestris formed a highly supported clade alongside C. olla, N. gilchristi, and M. melo.Buccinoidea, Muricoidea, Volutoidea, and Olivoidea clustered together to form a branch (bootstrap probability of 0.7459).Within this branch, the bootstrap support value for the relationship among Muricoidea, Volutoidea, and Olivoidea is 0.6916.

Basic Features of the Mitogenome of F. rupestris
Fulgoraria rupestris, like most gastropods, possesses a mitochondrial genome consisting of 37 genes [60][61][62].The sequence lengths of the other three species (M.melo, N. gregarious, and C. olla) range from 15,312 to 15,721 base pairs.In the mitochondrial genome of F. rupestris, there is a D-loop region spanning 975 base pairs in length between trnF and cox3, which results in a total mitochondrial genome length of 16,223 bp in F. rupestris.The genome composition exhibits a pronounced AT bias, consistent with findings reported in previous studies [63][64][65].
Mitochondrial genes, with their high conservation, limited recombination, and maternal inheritance, are utilized to elucidate the evolutionary relationships among various animal taxa [66,67].In contrast to vertebrate mitochondrial genomes, those found in mollusks demonstrate notable heterogeneity in both length and structure.This variability is attributed to disparities in gene loss or duplication, as well as variations in the position and strand specificity of tRNA, protein-coding, and rRNA genes [68].In this study, among the four known species sequences within Volutidae, three exhibit a consistent gene order, while M. melo lacks trnF.This deletion in the non−coding region may be attributed to slippage events occurring in regions with high A/T or AT/TA repeats.

Phylogenetic Analysis
The classification of Neogastropoda remains contentious.This study is consistent with the taxonomic research by Bouchet et al. [16], emphasizing the monophyly of Buccinoidea, Muricoidea, Volutoidea, and Olivoidea [69].Olivoidea, Volutoidea, and Muricoidea are closely related.This is consistent with the findings of Lemarcis et al. [70].This study follows the classification by Harasewych et al. [25] and Fedosov et al. [16].In the phylogenetic tree, Volutidae forms a clade, with F. rupestris being most closely related to N. gilchristiy.F. rupestris and N. gilchristiy exhibit greater morphological similarities.
Muricoidea, the second largest family in Neogastropoda, has not consistently exhibited monophyly in prior morphological and molecular studies [1,13,71,72].Barco et al. [73] conducted a study based on partial sequences of three mitochondrial genes (12S rRNA, 16S rRNA, and COI) and one nuclear gene (28S rRNA).Their analysis, employing Bayesian inference and maximum likelihood methods, supported the monophyly of Muricoidea [73].Previous morphological and molecular studies have not adequately validated the monophyly of Buccinoidea [1,13,71].Kantor et al. [74] found that Buccinoidea is monophyletic in Bayesian analysis but lacks support in ML analysis.Additionally, Galindo et al. confirmed Buccinoidea's monophyly, yet further investigation is required to validate this assertion [75].This study restored the monophyly of Buccinoidea, similar to the results of Oliverio et al., who used mitochondrial sequences (16S rRNA, 12S rRNA, and COI) for their analysis [72].Consistent with the study by Kantor et al., Austrosiphonidae and Tudiclidae are sister groups [74].Cominellina was originally a subfamily within Buccinidae.Kantor et al. found that Cominellina has no affinity with Buccinidae and is not included in any larger supported clusters within the core Buccinoidea [74].Therefore, it has been elevated to the rank of family and named Cominellidae.Our study has demonstrated this.In molecular phylogenetic investigations conducted by Puillandre et al. [76] and Yang et al. [77], Conoidea was identified as a monophyletic group, contrasting with the findings of our study.In our study, Conoidea is divided into three branches.This observation is consistent with the findings of Cunha et al. [13] and Zou et al. [21].The instability or conflicting branches observed within Conoidea may be attributed to the limited sampling of taxonomic units.
Although the number of species increased to 50 in this study, the internal phylogenetic relationship of Neogastropoda is still uncertain.Further comprehensive mitochondrial DNA sequencing of additional gastropod lineages is necessary to effectively address this issue.However, the rapid diversification at the origin of Neogastropoda and the complex evolutionary patterns of genes associated with morphological differentiation may also complicate phylogenetic inference [78].Therefore, additional nuclear sequence data need to be incorporated into phylogenetic analyses [13].Further research is required to elucidate the phylogenetic positions of each superfamily within Neogastropoda.

Conclusions
This study conducted a comprehensive analysis of the complete mitochondrial genome of F. rupestris using molecular biology methods.The analysis encompassed the examination of mitochondrial genome content, organization, codon usage, gene arrangement, phylogenetic relationships, and positive selection.Based on the complete mitochondrial genome sequences,

Figure 2 .
Figure 2. The secondary structure of F. rupestris mitochondrial tRNA.

Figure 2 .
Figure 2. The secondary structure of F. rupestris mitochondrial tRNA.

Figure 3 .
Figure 3. Amino acid composition in the mitochondrial genome of F. rupestris.Figure 3. Amino acid composition in the mitochondrial genome of F. rupestris.

Figure 3 .
Figure 3. Amino acid composition in the mitochondrial genome of F. rupestris.Figure 3. Amino acid composition in the mitochondrial genome of F. rupestris.Genes 2024, 15, x FOR PEER REVIEW 9 of 16

Figure 5 .
Figure 5. Analysis of the selection pressure of Volutidae; ka refers to non-synonymous substitution value, and ks refers to synonymous substitution value.

Figure 5 .
Figure 5. Analysis of the selection pressure of Volutidae; ka refers to non-synonymous substitution value, and ks refers to synonymous substitution value.

Figure 6 .
Figure 6.The black circles represent the mitochondrial gene sequences of the remaining 44 species.

Figure 6 .
Figure 6.The black circles represent the mitochondrial gene sequences of the remaining 44 species.

Figure 7 .
Figure 7.The phylogenetic tree, constructed based on the 13 PCGs of 50 Neogastropoda species, displays support values (BI, ML) for each node.(ML/BI; the range of ML support values is from 0 to 100, while the range of BI support values is from 0 to 1.)

Figure 7 .
Figure 7.The phylogenetic tree, constructed based on the 13 PCGs of 50 Neogastropoda species, displays support values (BI, ML) for each node.(ML/BI; the range of ML support values is from 0 to 100, while the range of BI support values is from 0 to 1.)

Table 1 .
List of species analyzed in this study and their GenBank accession numbers, with the newly sequenced Fulgoraria rupestris species marked with an asterisk (*).

Table 2 .
Organization of the mitogenome of F. rupestris.
3.2.Analysis of rRNA and tRNA in the F. rupestris Mitochondrial Genome